Uso terapéutico de anticuerpos de yema de huevo para la prevención de la iniciación de la infección por clostridium difficile
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2016
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es
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Universidad Andrés Bello
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Licencia CC
Licencia CC
Resumen
Clostridium difficile es un bacilo anaerobio estricto, Gram positivo y formador de endoesporas.
La infección por C. difficile (ICD) representa del 20 al 30% de las diarreas infecciosas
intrahospitalarias relacionadas al consumo de antibióticos. Las manifestaciones clínicas varían
desde cuadros de diarrea leve hasta una manifestación típica de colitis pseudomembranosa. A pesar
de que la tasa de mortalidad de las ICD alcanza un 5%, el mayor problema en el manejo de la ICD
es la recurrencia de la infección, la cual varía entre 20 a 25% después del primer episodio.
Actualmente, el tratamiento estándar contra la ICD a base de antibióticos (i.e., metronidazol y
vancomicina) solo elimina la forma vegetativa de C. difficile, por lo tanto, persiste el morfotipo de
espora el cual es resistente a antibióticos, químicos o péptidos antimicrobianos y al ataque por parte
de células fagocíticas. Su multirresistencia y su habilidad para persistir en el tracto colónico hace
que la espora sea esencial para la iniciación y recurrencia de la ICD, por lo tanto, se convierte en
un atractivo blanco terapéutico. Una alternativa estratégica es la implementación de inmunización
pasiva mediante la administración oral de anticuerpos. El uso de inmunoterapia oral, en particular
el uso de inmunoglobulinas de yema de huevo (IgY) contra esporas de C. difficile, permitiría
reducir las esporas en el tracto colónico y en consecuencia prevenir la iniciación de la ICD. Por
consiguiente, en este estudio se logró producir una fuerte respuesta humoral en las gallinas
inmunizadas con esporas de C. difficile, permitiendo obtener un elevado título, con una especificad
antígeno-anticuerpo dentro de los rangos descritos en literatura y que no poseen reactividad cruzada
contra la microbiota fecal murina, lo que se traduce en IgY altamente específicas para esporas de
C. difficile (IgY-Cds). Además, se demostró la capacidad de las IgY-Cds en disminuir la adherencia
de esporas a células epiteliales in vitro, sin embargo, éstas no disminuyeron la eficiencia de
germinación de esporas de C. difficile. Finalmente, se demostró la capacidad de las IgY-Cds en
reducir la persistencia de esporas en el tracto colónico, disminuyendo significativamente la
adherencia de éstas in vivo, lo que se tradujo en una reducción significativa de la gravedad e
incidencia de la diarrea previniendo la iniciación de la ICD en el modelo murino. En definitiva,
estos resultados sugieren que una terapia de IgY-Cds permitiría reducir la presencia de esporas en
el tracto colónico y en consecuencia prevenir la iniciación de la ICD.
Clostridium difficile is a strict anaerobic bacilli, Gram positive and endospores forming. C. difficile infection (CDI) account for 20 to 30% of nosocomial infectious diarrhea related to antibiotic use. Clinical manifestations vary from mild diarrhea to a typical manifestation of pseudomembranous colitis. Besides generating the colonic epithelium damage, the main problem in the management of the CDI is the recurrence of the infection, which varies between 20 to 25% after the first episode. Currently, standard treatments against CDI with antibiotics (i.e., metronidazole and vancomycin) only eliminate the vegetative form of C. difficile, therefore the morphotype spore which is resistant to antibiotics, antimicrobial peptides or chemicals and resist the attack by phagocytic cells. Its multiresistance and ability to persist in the colonic tract makes spores essential for the initiation and recurrence of CDI and becomes an attractive therapeutic target. An alternative strategy is to implement passive immunization by oral administration of antibody. The use of oral immunotherapy, particularly the use of egg yolk immunoglobulin (IgY) against C. difficile spores, would reduce the spores in the colonic tract and thereby prevent initiation of the CDI. Therefore, in this study we were able to produce a strong humoral response in the hens immunized with C. difficile spores, allowing to obtain a high titer, an antigen-antibody specificity within the ranges described in literature and no cross-reactivity against murine fecal microbiota, which results in highly specific IgY for C. difficile spores (IgY-Cds). Furthermore, the ability of IgY-Cds in reducing spore adhesion to epithelial cells was demonstrated in vitro, although, IgYCds did not decrease the efficiency of germination of C. difficile spores. Finally, the ability of IgYCds in preventing spore persistence in the colonic tract was shown, which significantly reduced the adhesion of the spores in vivo, leading to a significant reduction in the severity and incidence of the diarrhea, thus preventing the initiation of the CDI in the mouse model. Taken together, these results suggest that IgY-Cds therapy would reduce the presence of spores in the colonic tract and thereby combat the initiation of the CDI.
Clostridium difficile is a strict anaerobic bacilli, Gram positive and endospores forming. C. difficile infection (CDI) account for 20 to 30% of nosocomial infectious diarrhea related to antibiotic use. Clinical manifestations vary from mild diarrhea to a typical manifestation of pseudomembranous colitis. Besides generating the colonic epithelium damage, the main problem in the management of the CDI is the recurrence of the infection, which varies between 20 to 25% after the first episode. Currently, standard treatments against CDI with antibiotics (i.e., metronidazole and vancomycin) only eliminate the vegetative form of C. difficile, therefore the morphotype spore which is resistant to antibiotics, antimicrobial peptides or chemicals and resist the attack by phagocytic cells. Its multiresistance and ability to persist in the colonic tract makes spores essential for the initiation and recurrence of CDI and becomes an attractive therapeutic target. An alternative strategy is to implement passive immunization by oral administration of antibody. The use of oral immunotherapy, particularly the use of egg yolk immunoglobulin (IgY) against C. difficile spores, would reduce the spores in the colonic tract and thereby prevent initiation of the CDI. Therefore, in this study we were able to produce a strong humoral response in the hens immunized with C. difficile spores, allowing to obtain a high titer, an antigen-antibody specificity within the ranges described in literature and no cross-reactivity against murine fecal microbiota, which results in highly specific IgY for C. difficile spores (IgY-Cds). Furthermore, the ability of IgY-Cds in reducing spore adhesion to epithelial cells was demonstrated in vitro, although, IgYCds did not decrease the efficiency of germination of C. difficile spores. Finally, the ability of IgYCds in preventing spore persistence in the colonic tract was shown, which significantly reduced the adhesion of the spores in vivo, leading to a significant reduction in the severity and incidence of the diarrhea, thus preventing the initiation of the CDI in the mouse model. Taken together, these results suggest that IgY-Cds therapy would reduce the presence of spores in the colonic tract and thereby combat the initiation of the CDI.
Notas
Tesis (Magister en Biotecnología)
Este trabajo fue elaborado bajo la supervisión del Director de Tesis Dr. Daniel Paredes-Sabja, en el laboratorio Gut Microbiota and Clostridia Research Group, Universidad Andrés Bello, aprobado por los miembros de la Comisión de evaluación
Este trabajo fue elaborado bajo la supervisión del Director de Tesis Dr. Daniel Paredes-Sabja, en el laboratorio Gut Microbiota and Clostridia Research Group, Universidad Andrés Bello, aprobado por los miembros de la Comisión de evaluación
Palabras clave
Yema de Huevo, Uso Terapéutico, Anticuerpos, Clostridium Difficile